On the nature of leukotriene C4 synthase in human platelets.

Leukotriene C4 is considered to play a major role in several important pathophysiological conditions, e.g., allergy, asthma, and shock. The present investigation demonstrates the presence in human platelets of a membrane-associated enzyme catalyzing the final step in the biosynthesis of leukotriene C4. This leukotriene C4 synthase was shown to be distinct from previously characterized "microsomal" and soluble glutathione transferases. The latter enzymes did not contribute significantly to the leukotriene A4 conjugating activity in platelets. As determined with leukotriene C4 synthase of a crude membrane fraction from human platelets, the Km value was 7 microM and the V value was 0.56 nmol x min-1 x mg-1 with leukotriene A4 as substrate. The enzyme was 20-fold more efficient with leukotriene A4 than with leukotriene A5 and 30-fold more efficient than with the unphysiological derivative leukotriene A4 methyl ester, as measured by the corresponding V/Km values; 14,15-leukotriene A4 was not a substrate. Platelets should be a useful source for the purification and further characterization of human leukotriene C4 synthase.     

Covalent binding properties of the C4A and C4B isotypes of the fourth component of human complement on several C1-bearing cell surfaces

In a previous study we demonstrated that the thioester-mediated transacylation of the human C4B isotype onto sheep erythrocytes (ES) was approximately fourfold more efficient than that of C4A. Moreover, although C4B formed predominantly ester linkages, C4A displayed a preference for amide bond formation. We therefore suggested that the relative functional activity observed for the two isotypes would be a combined reflection of their nucleophilic preference and the surface composition of the C1-bearing target. The present study tests this hypothesis. Chemical modification of amino groups on Es with ethylacetimidate produced a twofold decrease in the C1-dependent binding of C4A isotype, while having a negligible effect on C4B binding. Furthermore, with human erythrocytes and two human leukocyte cell lines, K562 and U937, the C4B to C4A deposition ratio decreased from greater than 4 with ES to between 1.5 and 2. Irrespective of the target, C4A and C4B maintained their preference for forming amide and ester bonds, respectively. Interestingly, SDS-PAGE profiles of radiolabeled C4A and C4B, which had been covalently deposited on the various cells, suggested a further degree of transacylation specificity, as the two isotypic alpha-chains sometimes bound to different membrane components. These differences were not easily accounted for by simple differences in the abundance of the preferred nucleophile for each isotype on a given surface constituent, nor were they due to the preferential binding of one isotype to the sensitizing antibody. We speculate that nascent C4B may contain a substrate binding site that facilitates productive attack on the thioester carbonyl by molecules containing the class of nucleophile preferred by each isotype.     

Genetic polymorphism of human C4-binding protein

Two different forms of human C4-bp, C4-bp A and C4-bp B, have been identified by isoelectric focusing (IEF) of neuraminidase-treated EDTA-plasma samples. Family studies demonstrate Mendelian segregation of these forms, indicating that they are under gentic control. This conclusion is supported by IEF analysis of the two variants purified by affinity chromatography. Under completely denaturing conditions, C4-bp B was found to be composed of two subunits that focused at different pH, whereas C4-bp A contains only the more basic one. These results suggest that a single autosomal locus with at least two codominant alleles coding for the subunits controls the IEF variation of C4-bp in humans. The allele designated C4BP*1 codes for a subunit that, after neuraminidase treatment, focuses at pH = 6.65. The allele C4BP*2 codes for a different subunit that focuses at pH = 6.60. The C4-bp A phenotype corresponds to the genotype C4BP*1,C4BP*1 and the phenotype C4-bp B to the genotype C4BP*1,C4BP*2. The phenotype corresponding to the C4BP*2,C4BP*2 homozygous genotype has not been encountered thus far. Initial linkage data indicate that the C4BP locus is not closely linked to either the HLA or to the C3 loci.     

The low C5 convertase activity of the C4A6 allotype of human complement component C4.

We have compared the C5-convertase-forming ability of different C4 allotypes, including the C4A6 allotype, which has low haemolytic activity and which has previously been shown to be defective in C5-convertase formation. Recent studies suggest that C4 plays two roles in the formation of the C5 convertase from the C3 convertase. Firstly, C4b acts as the binding site for C3 which, upon cleavage by C2, forms a covalent linkage with the C4b. Secondly, C4b with covalently attached C3b serves to form a high-affinity binding site for C5. Purified allotypes C4A3, C4B1 and C4A6 were used to compare these two activities of C4. Covalently linked C4b-C3b complexes were formed on sheep erythrocytes with similar efficiency by using C4A3 and C4B1, indicating that the two isotypes behave similarly as acceptors for covalent attachment of C3b. C4A6 showed normal efficiency in this function. However, cells bearing C4b-C3b complexes made from C4A6 contained only a small number of high-affinity binding sites for C5. Therefore a lack of binding of C5 to the C4b C3b complexes is the reason for the inefficient formation of C5 convertase by C4A6. The small number of high-affinity binding sites created, when C4A6 was used, were tested for inhibition by anti-C3 and anti-C4. Anti-C4 did not inhibit C5 binding, whereas anti-C3 did. This suggests that the sites created when C4A6 is used to make C3 convertase may be C3b-C3b dimers, and hence the low haemolytic activity of C4A6 results from the creation of low numbers of alternative-pathway C5-convertase sites.     

A single arginine to tryptophan interchange at beta-chain residue 458 of human complement component C4 accounts for the defect in classical pathway C5 convertase activity of allotype C4A6. Implications for the location of a C5 binding site in C4.

In general, C4A allotypes of human C4 show one-fourth to one-third the hemolytic activity of C4B allotypes. An exception to this rule is C4A6 which is almost totally deficient in hemolytic activity. Previous studies have localized the defect in C4A6 to the C5 convertase stage. Of the two critical events required for C5 cleavage, namely formation of a covalent adduct between C3b and the C4b subunit of the C3 convertase (C4b2a), and binding of C5 to this C4b-C3b complex, it is a defect in the latter step that accounts for the aberrant activity of C4A6. DNA sequencing studies described in a companion paper have suggested that the sole C4A6-specific difference was a Trp for Arg replacement at beta-chain residue 458. To directly ascertain whether this single substitution was responsible for the hemolytic defect in C4A6, we have used site-directed mutagenesis to introduce this change into both C4A and C4B cDNA expression plasmids. We found that the R to W replacement totally abrogated hemolytic activity. However, irrespective of the amino acid at residue 458, the mutant proteins behaved like their wild-type counterparts with respect to covalent binding to C1-bearing targets, i.e., the C4B recombinants displayed higher binding to sheep and human red cells than did the C4A counterparts. Furthermore, the mutants were able to form covalent C4b-C3b adducts. There was, however, substantially less C5 cleavage produced by cell-bound C4boxy23b complexes made with R458W mutant C4B than with wild-type C4B. These results are consistent with the sole defect in the mutants being at the C5 binding stage and strongly suggest that Arg 458 of the C4 beta-chain contributes to the C5 binding site of the molecule.     

Murine C4-binding protein: a rapid purification method by affinity chromatography

A simple, 3-step method was described for purification of murine C4 binding protein (C4-bp), a recently recognized serum protein that functions as one of the regulatory proteins of the complement system. The method consists of 1) affinity chromatography using TNBS-BGG-conjugated Sepharose beads, 2) gel filtration on a Sepharose 6B column, and 3) heparin-Sepharose chromatography. By this method, milligram quantities of C4-bp can be easily purified by more than 500-fold from EDTA-serum of various mouse strains, and the whole purification process can be completed within 1 wk. The overall yield of C4-bp is about 15%. The C4-bp thus prepared is homogeneous as judged by SDS-polyacrylamide gel electrophoresis and immunelectrophoresis. The purified mouse C4-bp showed physicochemical properties very similar to those described for human C4-bp. Like human C4-bp, mouse C4-bp is composed of several apparently identical subunits of the m.w. of 80,000. However unlike the human counterpart, the subunits of mouse C4-bp are not linked by disulfide bonds but are connected by non-covalent forces that can be disrupted by SDS. The purified mouse C4-bp retained binding affinity for C4 and showed unaltered antigenicity. Immunization of rabbits with the purified mouse C4-bp resulted in the production of potent and monospecific antisera.     

Copy number analysis of complement C4A, C4B and C4A silencing mutation by real-time quantitative polymerase chain reaction

Low protein levels and copy number variation (CNV) of the fourth component of human complement (C4A and C4B) have been associated with various diseases. High-throughput methods for analysing C4 CNV are available, but they commonly do not detect the most common C4A mutation, a silencing CT insertion (CTins) leading to low protein levels. We developed a SYBR? Green labelled real-time quantitative polymerase chain reaction (qPCR) with a novel concentration range approach to address C4 CNV and deficiencies due to CTins. This method was validated in three sample sets and applied to over 1600 patient samples. CTins caused C4A deficiency in more than 70% (76/105) of the carriers. Twenty per cent (76/381) of patients with a C4A deficiency would have been erroneously recorded as having none, if the CTins had not been assessed. C4A deficiency was more common in patients than a healthy reference population, (OR?=?1.60, 95%CI?=?1.02-2.52, p?=?0.039). The number of functional C4 genes can be straightforwardly analyzed by real-time qPCR, also with SYBR? Green labelling. Determination of CTins increases the frequency of C4A deficiency and thus helps to elucidate the genotypic versus phenotypic disease associations.     

Conversion of leukotrienes A4 to C4 in cell-free systems

A procedure for assaying leukotriene C4 synthase activity in cell-free extracts has been presented. Leukotriene A4 methyl ester was as active a substrate as leukotriene A4 (Na salt) for the synthesis. The methyl ester is the substrate of choice, because (1) it is more stable than the sodium salt, (2) it is not a substrate of epoxide hydrolase for leukotriene B4 synthesis, and (3) it gives a lower blank than an equimolar concentration of leukotriene A4. The enzyme activity in rat liver, guinea pig and human lungs, and human nasal polyp was chiefly membrane-bound, although the cytosol contained some activity.     

Resolving the compartmentation and function of C4 photosynthesis in the single-cell C4 species Bienertia sinuspersici

Bienertia sinuspersici is a land plant known to perform C(4) photosynthesis through the location of dimorphic chloroplasts in separate cytoplasmic domains within a single photosynthetic cell. A protocol was developed with isolated protoplasts to obtain peripheral chloroplasts (P-CP), a central compartment (CC), and chloroplasts from the CC (C-CP) to study the subcellular localization of photosynthetic functions. Analyses of these preparations established intracellular compartmentation of processes to support a NAD-malic enzyme (ME)-type C(4) cycle. Western-blot analyses indicated that the CC has Rubisco from the C(3) cycle, the C(4) decarboxylase NAD-ME, a mitochondrial isoform of aspartate aminotransferase, and photorespiratory markers, while the C-CP and P-CP have high levels of Rubisco and pyruvate, Pidikinase, respectively. Other enzymes for supporting a NAD-ME cycle via an aspartate-alanine shuttle, carbonic anhydrase, phosophoenolpyruvate carboxylase, alanine, and an isoform of aspartate aminotransferase are localized in the cytosol. Functional characterization by photosynthetic oxygen evolution revealed that only the C-CP have a fully operational C(3) cycle, while both chloroplast types have the capacity to photoreduce 3-phosphoglycerate. The P-CP were enriched in a putative pyruvate transporter and showed light-dependent conversion of pyruvate to phosphoenolpyruvate. There is a larger investment in chloroplasts in the central domain than in the peripheral domain (6-fold more chloroplasts and 4-fold more chlorophyll). The implications of this uneven distribution for the energetics of the C(4) and C(3) cycles are discussed. The results indicate that peripheral and central compartment chloroplasts in the single-cell C(4) species B. sinuspersici function analogous to mesophyll and bundle sheath chloroplasts of Kranz-type C(4) species.     

The C4A and C4B isotypic forms of human complement fragment C4b have the same intrinsic affinity for complement receptor 1 (CR1/CD35)

Several previous reports concluded that the C4b fragment of human C4A (C4Ab) binds with higher affinity to CR1 than does C4Bb. Because the isotypic residues, (1101)PCPVLD and (1101)LSPVIH in C4A and C4B, respectively, are located within the C4d region, one may have expected a direct binding contribution of C4d to the interaction with CR1. However, using surface plasmon resonance as our analytical tool, with soluble rCR1 immobilized on the biosensor chip, we failed to detect significant binding of C4d of either isotype. By contrast, binding of C4c was readily detectable. C4A and C4B, purified from plasma lacking one of the isotypes, were Cs converted to C4Ab and C4Bb. Spontaneously formed disulfide-linked dimers were separated from monomers and higher oligomers by sequential chromatographic steps. The binding sensorgrams of C4Ab and C4Bb monomers as analytes reached steady state plateaus, and these equilibrium data yielded essentially superimposable saturation curves that were well fit by a one-site binding model. Although a two-site model was required to fit the equilibrium-binding data for the dimeric forms of C4b, once again there was little difference in the K(D) values obtained for each isotype. Independent verification of our surface plasmon resonance studies came from ELISA-based inhibition experiments in which monomers of C4Ab and C4Bb were equipotent in inhibiting the binding of soluble CR1 to plate-bound C4b. Although divergent from previous reports, our results are consistent with recent C4Ad structural data that raised serious doubts about there being a conformational basis for the previously reported isotypic differences in the C4b-CR1 interaction.     

Stimulation of HLA-A,B,C by IFN-alpha. The derivation of Molt 4 variants and the differential expression of HLA-A,B,C subsets.

Spontaneous mutants with altered HLA-A,B,C response to interferon-alpha (IFN-alpha) were isolated from the human thymus leukemia cell line Molt 4. Using fluorescein isothiocyanate (FITC)-conjugated W6/32 (a monoclonal antibody to HLA-A,B,C) and the fluorescence-activated cell sorter, the cells with highest and lowest fluorescence after 24-48 h of IFN-alpha treatment were selected and expanded. After several cycles of selection, mutant clones with low (greater than 10% of wild-type) and high (three times better) response were obtained. A similar protocol was employed to derive high responder mutants with the monoclonal antibody YT76, which recognises a subset of HLA strongly induced by IFN-alpha. Stable clones were derived for which YT-HLA induction was 7-fold that of Molt 4 cells and for which HLA induction occurred at 100-fold lower concentrations of IFN-alpha. The high response phenotype of the mutants was not accompanied by a significant increase in the constitutive level of expression of HLA-A,B,C (in the absence of IFN). The increase in the level of HLA-A,B,C expression after IFN-alpha treatment is mostly accounted for by the increase in the expression of a subset of HLA molecules, detected by the monoclonal antibody YT76 including HLA-B molecules.     

Deficiencies in C4A and C4B isotypes of human complement revealed by isoelectrofocusing and chemical reactivity of activated forms

An approach is proposed to detect deficiencies in isotypes A and B of the C4 component of human complement, based on the calculation of the ratio of their IEA activities and the ratio of their quantities determined by isoelectrofocusing of their desialated forms with chemiluminescent detection in an immunoblot. The ratios of the quantities and activities of C4A/C4B practically coincided when determined in blood serum of 20 patients, many of which had inherited deficiencies in the C4 component isotypes.     

Deficiencies in C4A and C4B isotypes of human complement revealed by isoelectrofocusing and chemical reactivity of activated forms

An approach is proposed to detect deficiencies in isotypes A and B of the C4 component of human complement, based on the calculation of the ratio of their IEA activities and the ratio of their quantities determined by isoelectrofocusing of their desialated forms with chemiluminescent detection in an immunoblot. The ratios of the quantities and activities of C4A/C4B practically coincided when determined in blood serum of 20 patients, many of which had inherited deficiencies in the C4 component isotypes.     

Deletion of complement C4 and steroid 21-hydroxylase genes in the HLA class III region

Molecular maps have been prepared of the HLA region on human chromosome 6 that includes the complement C4 and steroid 21-hydroxylase genes (21-OH), using DNA of individuals deficient (QO) in either of the two forms C4A or C4B. In all, 18 haplotypes with C4A QO were examined by Southern analysis and two had deletions of 28-30 kb that included both the C4A and 21-OHA genes. Of six C4B QO haplotypes, one had a deletion that included both the C4B and 21-OHA genes. Thus, some of the C4 null alleles are due to deletion of the gene but the majority in this sample are not. Deletion occurred in two common haplotypes suggesting that in the population as a whole, C4A deficiency is due to deletion in about one-half the C4A QO haplotypes. As duplication of C4A or C4B genes does occur, the possibility that unequal cross-over could explain the C4 deletion was examined by preparing cosmid clones from the DNA of an individual typed C4A QO. A cloned genomic fragment containing the single C4B gene was isolated and found to be similar to the homologous region of a cosmid from a normal individual carrying a C4A gene. This suggests that if a cross-over has occurred it is in a region where the two genes are identical. The biological significance of the rather frequent occurrence in the population of haplotypes with C4A or C4B deletion together with the accompanying deletion of the 21-OHA gene is discussed.     

Isotyping of human C4 complement using differences in the functional activity of C4A and C4B isotypes

The difference in the functional activity of the isotypes A and B of component C4 of human complement was used to determine their ratio and to detect the inherited deficiency of the isotypes. ELISA methods were developed for the quantitative assay of component C4 (conventional sandwich method) and its functional activity. When determining the functional activity, the classic pathway of the complement and therefore of component C4 was activated by activators sorbed on ELISA microplates (immunoglobulin IgG3 or liposaccharide of the Shigella sonnei cell walls, which activates the complement by binding component C1). The nascent fragment C4b is covalently bound to the target activator; C4Ab binds better to the target protein (immunoglobulin), and C4Bb to the target carbohydrate (liposaccharide). Therefore, when immunoglobulin is a target activator, isotype C4A is bound and determined; and when the complement is activated by liposaccharide, isotype C4B is determined. The ratio of the activities determined by the two methods indicates a deficiency in the individual isotypes of component C4 or its absence. The rabbit polyclonal monospecific antibodies against the human component C4 and the conjugates of these antibodies with horseradish peroxidase were used in the methods described.     

Inhibition of the covalent binding reaction of complement component C4 by penicillamine, an anti-rheumatic agent.

D(-)-Penicillamine [D(-)-beta beta-dimethylcysteine] is an anti-arthritic drug, but its use is limited by adverse side effects, which include problems in immune-complex clearance. Complement is important as a source of inflammatory mediators in rheumatoid arthritis and is also involved in immune-complex clearance. Thus inhibition of the complement cascade would be likely to contribute to both the therapeutic and the toxic effects of penicillamine. It is shown that penicillamine and cysteine are potent inhibitors of the covalent binding of activated complement component C4 to immune complexes. [35S]Cysteine itself becomes covalently bound to C4b through the thioester site. Penicillamine and cysteine are more reactive with the C4A isotype than with the C4B isotype of the HLA class III protein C4. The limited amino acid sequence differences between C4A and C4B include a cysteine/serine interchange, and it is suggested that the cysteine residue in C4A contributes to the increased rate of reaction of C4A with the alpha-amino-beta-thiol compounds.     

Covalent binding properties of the human complement protein C4 and hydrolysis rate of the internal thioester upon activation.

The complement proteins C3 and C4 have an internal thioester. Upon activation on the surface of a target cell, the thioester becomes exposed and reactive to surface-bound amino and hydroxyl groups, thus allowing covalent deposition of C3 and C4 on these targets. The two human C4 isotypes, C4A and C4B, which differ by only four amino acids, have different binding specificities. C4A binds more efficiently than C4B to amino groups, and C4B is more effective than C4A in binding to hydroxyl groups. By site-directed mutagenesis, the four residues in a cDNA clone of C4B were modified. The variants were expressed and their binding properties studied. Variants with a histidine residue at position 1106 showed C4B-like binding properties, and those with aspartic acid, alanine, or asparagine at the same position were C4A-like. These results suggest that the histidine is important in catalyzing the reaction of the thioester with water and other hydroxyl group-containing compounds. When substituted with other amino acids, this reaction is not catalyzed and the thioester becomes apparently more reactive with amino groups. This interpretation also predicts that the stability of the thioester in C4A and C4B, upon activation, will be different. We measured the time course of activation and binding of glycine to C4A and C4B. The lag in the binding curve behind the activation curve for C4A is significantly greater than that for C4B. The hydrolysis rates (k0) of the thioester in the activated proteins were estimated to be 0.068 s-1 (t1/2 of 10.3 s) for C4A and 1.08 s-1 (t1/2 of 0.64 s) for C4B. These results indicate that the difference in hydrolysis rate of the thioester accounts, at least in part, for the difference in the binding properties of C4A and C4B.